DESCRIPTION (adapted from the application) This research project is designed to provide the applicant, George Vassilopoulos, with the necessary scientific expertise for an independent career in academic medicine and clinically oriented research in the fields of gene transfer technology and stem cell biology. The aim of this proposal is to investigate whether vectors based on the human foamy virus (HFV) can transduce murine hematopoietic stem cells (HSC). HFV are considered as nonpathogenic to humans and have the potential to transduce quiescent cells, both useful features for HSC gene therapy. Gene transfer into HSC can benefit trials where genetic modification of HSCs is highly desirable. In this proposal, HFV vectors will be tested in murine bone marrow transplantations with genetically modified cells as a preclinical model. Gene transfer will be assayed both at the protein and DNA level. Specific Aim 1 deals with the construction of foamy vectors with: a) readily assayable reporter genes like the neo and enhanced green fluorescence protein (EGFP), b) MLV-LTR-based internal promoters which are thought to perform better in HSCs and c) a therapeutic gamma-globin cassette. Expression from these vectors will be tested in transplanted animals. Specific Aim 2 is directed into developing a PCR assay to quantitate the levels of vector DNA in the peripheral blood of the transplanted mice. Stem cell transduction events will be investigated by an inverse PCR assay that can detect the vector integration sites in the different hematopoietic lineages. Specific Aim 3 is oriented into developing optimal conditions for the transduction of murine bone marrow cells with the HFV vectors. Specifically we will investigate a) the potential use of adhesion molecules during transduction, b) the 5FU conditioning, the two days prestimulation step and the extent of ex-vivo transduction of the donor cells, c) the expression of vectors developed in Aim 1 in murine bone marrow transplantation studies employing the optimal transduction schemes developed in subaims a and b, d) whether preselection schemes with the EGFP vector can improve gene marking rates and e) the expression of the HFV vectors in secondary transplants from long term expressing mice Specific Aim 4 investigates the possible risks associated with wild type HFV infection of HSCs. Although current vector stocks are free of wild type foamy virus, there is a remote but distinct possibility for generation of a replication competent retrovirus. We plan to infect murine bone marrow with wild type HFV, transplant the infected cells and follow the mice for signs of disease, monitor the viral load by PCR and the immune response by serological assays as a preclinical safety study.